The potential role of CD8+, cytolytic T lymphocytes [CTLs] in resistance to infectious and malignant diseases has been emphasized by recent developments in immunology. Antigen-specific CTLs are recognized as a possible defense mechanism in infection with HIV-1 (1-3), cytomegalovirus (4), and in malaria (5). Antigens that are recognized by melanoma-specific CTLs also have been identified by Boone and colleagues (6,7). These studies document the specificity of CTLs that recognize clinically important targets. Less is understood about the initial generation of these CTLs, however.
As in most T cell responses, the precursors for active CTLs are quiescent lymphocytes that must be induced to expand clonally and develop effector functions. For CTL activation to occur, not only must antigens be presented as peptide fragments on MHC products, but the antigen-MHC complexes must also be introduced on cells with the requisite accessory functions that lead to T cell growth and cytolytic activity. Studies of killer cells response to transplantation antigens, provide evidence that an effective way to induce human CTLs is to present antigens on dendritic cells (8). Dendritic cells are specialized accessory cells for the initiation of many T cell dependent immune responses [reviewed in (9)].
T cell receptors on CD8.sup.+ T cells recognize a complex consisting of an antigenic peptide, .beta.-2 microglobulin and Class I major histocompatibility complex (MHC) heavy chain (HLA-A, B, C, in humans). Processing and presenting of peptides on dendritic cells involves digesting of endogenously synthesized proteins and transporting them into the endoplastic reticulum, bound to Class I MHC heavy chain and .beta.2 microglobulin, and finally expressing the digested peptide in the cell surface in the groove of the Class I MHC molecule. Therefore, T cells can detect molecules that originate from proteins inside cells, in contrast to antibodies that detect intact molecules expressed on the cell surface. Consequently, CD8+ CTL are able to kill clinically important targets such as virus infected cells, tumors, and certain tissues attacked during autoimmune diseases.
Dendritic cells are potent antigen presenting cells for several immune responses. When exposed to replicating influenza virus, mouse dendritic cells stimulate strong cytolytic responses from CD8+ T cells. Prior work had not identified underlying mechanisms particularly the efficiency with which influenza virus infect dendritic cells, and whether the dendritic cells remain viable after exposure to influenza. Accordingly, it would be desirable to identify mechanisms to deliver antigen on dendritic cell Class I molecules for presentation to CD8+ CTLs.
The proficiency of dendritic cells as APCs has been attributed to their ability to aggregate antigen responsive T cells into clusters, high expression of MHC Class I and Class II molecules, as well as adhesion and co-stimulatory molecules, and efficient endocytic activity for the MHC Class II pathway. Dendritic cells can sensitize quiescent human T cells when few MHC Class II molecules are occupied by antigen [a maximum of 0.1% of surface MHC Class II molecules or 2000 molecules], indicating that low levels of signal when presented on dendritic cells, are sufficient to generate T cell responses. However, the efficiency with which dendritic cells handle Class I restricted antigens is not known, one difficulty being the need to identify dendritic cell antigens which are Class I.
The development of new strategies in immunotherapy for treatment of cancers and pathogens is greatly needed. In particular, improved mechanisms for prophylaxis and therapy are needed in influenza, since control of the respiratory infection is not readily achieved through current approaches to vaccination. For example, presently available vaccines are not designed to induce killer cells but instead boost antibody responses to viral antigens that undergo antigenic drift and shift (10). It is known that dendritic cells are a component of the alveolar septae and airway epithelium of the lung (11,12), and that the appearance of influenza virus-specific CTLs is associated with a more rapid clearance of virus from nasal washings (13).
Influenza virus is also an agent used to dissect the different pathways for antigen presentation of dendritic cells and analyze the specificity of CTLs. Townsend et al. reported that viral proteins were processed in the dendritic cell cytoplasm and presented as peptides in association with Class I MHC products of the infected cell (14,15). Morrison et al. used influenza virus to distinguish two pathways for antigen presentation to CTLs (16). One emanates from acidic endocytic vesicles and leads to presentation on MHC Class II to CD4+ CTLs; the other emanates from a nonacidic biosynthetic compartment for presentation on MHC Class I to CD8+ CTLs.
There is considerable evidence, primarily in mouse cell cultures, that dendritic cells effectively present viral antigens to T cells (26,28-30). Murine dendritic cells can be infected by influenza virus and elicit potent CTL responses (26,29). The responses are dependent upon the synthesis of endogenous viral proteins (26). Efforts to extend these results to humans have been unpredictable.
CTLs that have been the subject of investigation in humans are usually generated from unseparated blood mononuclear cells and/or repeated stimulation of responding lymphocytes with exogenous IL-2 and viral antigens (4,5,13,14,17-22). For example, Biddison et al (17) used repeatedly stimulated human blood cells in their elegant mapping studies of influenza peptides that are presented to CTLs. An efficient and effective system to generate human antigen specific cyntoxic T cells in general, and in particular influenza specific killer cells needs to be identified, especially one that capitalizes on the efficient accessory function of dendritic cells.
The use of dendritic cells to process and present antigens is described in Steinman application PCT/US93/03141. Accordingly, it would be useful to provide additional means of delivering various antigens to specific populations of dendritic cells. Such delivery systems could be useful for providing an efficient and effective system to generate human antigen specific cytotoxic T cells in general and, in particular, influenza specific killer cells.
Influenza A virus infection remains a major cause of mortality and morbidity, primarily because the control of the respiratory illness has not been achieved through vaccination. Current vaccines are designed to boost antibody responses to viral antigens [HA and NA] that undergo antigenic drift and shift (10). Consequently, the protective effects of antibodies decline with time. Vaccines directed towards the induction of influenza virus-specific cytotoxic T cell immune responses might be far more effective, since CTL have been shown to express cross reactivity in recognition of subtypes of influenza A (18). There is evidence in humans that CTL responses play a role in recovery from infection. McMichael et al (13) related levels of CTL immunity to clearance of nasal virus by normal donors inoculated with live virus. A clear association was observed between CTL responses and clearance of virus.
An ideal vaccine would utilize cross-reactive antigens, induce CD8+ CTL responses in most hosts, and have an efficient means of delivery. Several approaches to induce CTLs with these properties have been attempted in a number of systems. They include delivery of Class I-restricted peptides with adjuvant (58-61), conjugated to lipid (62), complexed with immune stimulating complexes (ISCOMs) (63), or inserted into liposomes (64, 65). The injection of DNA encoding the immunizing antigen directly into skeletal muscle (66) has also been reported to induce CTL.
Until recently, dendritic cells have not been directly considered in strategies to design new vaccines that generate CD8+ CTLs. Targeting antigen to dendritic cells has several advantages; one can maximize the efficiency of T cell activation (9), and avoid anergy induction (67) or the use of adjuvants (68). For example, dendritic cells pulsed with antigen in vitro and delivered in vivo to mice have been highly effective for generating CD4+ immune responses to protein antigens and microbes (68, 69). Mouse dendritic cells pulsed with Class I restricted peptides of NP (26), HIV peptides (70), or given antigen via pH sensitive liposomes (65) into the cytoplasm can induce CTL responses.